Polyhydroxy steroids and processes for their preparation



United States Patent ABSTRACT OF THE DISCLOSURE Synthesis of the insect and crustacean hormone, crustecdysone, novel polyhydroxy steroids and novel intermediates for the preparation thereof.

The present invention relates to novel cyclopentanopolydrophenanthrene compounds and to processes for their preparation. p

More particularly the invention relates to the total synthesis of the insect and crustacean steroid hormone,

crustecdysone, and related compounds from known starting compounds as well as to the novel intermediate compounds useful in the synthesis thereof.

Crustecdysone is a crustacean moulting hormone which differs from the known insect hormone ecdysone by the presence of an additional hydroxy group at C-20.

The novel compounds of the present invention which are useful for the synthesis of Crustecdysone and anal ogues thereof affect the growth of crustaceans and insects by controlling skin moults, that is, they cause ecdysis or skin shedding and stimulate maturation at all pre-adult stages of life. Thus they are useful in managing and controlling the population of crustaceans and insects. Crustecdysone and the novel compounds of the present invention which are employed in the same manner as the. related known insect hormone ecdysone and which may be employed in a range of from 0.001 microgra'm to about 20 micrograms per insect, are represented by the following formulas:

3,481,926 Patented Dec. 2, 1969 ice In the above formulas R represents u-hydrogen or B- hydrogen; R represents hydrogen or hydroxy; R and R are each hydrogen or a hydrocarbon carboxylic acyl group of less than 12 carbon atoms, or taken together,

The presence of asymmetric carbons in the various positions on the above formulas permit the existence of visomeric forms. As hereinafter set forth, the processes of the present invention generate both forms as stated and these forms are readily separated from one another by the conventional methods of chromatography, fractional crystallization, and the like. The present invention embraces all such isomeric forms. The use of the wavy line, .5, indicates both'; alpha and-beta configurations either singularly or collectively. With specific reference to the configuration at C-20 and C-22, the designations cc and ,8 are used in accordance with the Fischer convention. The acyl group is derived from hydrocarbon .carboxylic 'acids containing less than 12 carbon a'toms'which may 3,481,926 3 The preparation of crustecdysone and the novel com pounds of the present invention may be illustrated by the following equation:

CHzOH @Reductlon B 0 I @CrOa-Pyridine B 0 E E R I R H loxidation Sodium acetate OH 0H CH0 x i KHCO: X I

B 0 aqueous methanol B @dihydropyran t! @SeOa m H I 0 go \0/ CH0 CH1 CHO o- A o- R! 0- B o VIII:R'==H VII:R=H IXa:R'=OH l Grignard lGrignard OR! OR7 CH R CH i 0R1 CH3 3 CH;

\ /H A/ \l/ |\OR7 I 0R7 CH3 CH5 7 i A O I I i A O l B 0 i B 0- E u u 0 X 0 XI K2C O3, aq. methanol @KzC 0a, aq. methanol @Hydrogenetion @Hydrogenation @Acid @Acid OH (PH H OH OH OH i 0H 3 i on /l\ \i/N OH OH CH3 CH3 l I- nol H i it 110- HO ll ll 0 XII O XIV 9 CH 5 OH 3 l CH3 L!I orn OH OH CH3 C 5 HO I/ I t, HO XIII y) XV In the above formulas, A and B have the same meaning as heretofore set forth, R is keto or hydroxy and R is the tetrahydropyran-Z-yl group.

In practicing the process outlined above, the starting compound is produced from known compounds. More specifically, 25,35 diacetoxy 5oz hydroxy 6 keto 22, 23 bisnorchol 7 en 24 oic acid methyl ester described by Siddall et a1, JACS 88, 379-380 (1966), is acetylated in the presence of an acid catalyst such as ptoluenesulfonic acid to obtain the corresponding triacetate, i.e., 2fl,3}3,5a triacetoxy 6 keto 22,23 bisnorchol 7 en 24 oic acid methyl ester. The tertiary acyloxy group is removed by treatment with strongly acidic chromous chloride with the result that the thermodynamically less stable Sa-hydrOgen is formed to yield the known compound 25,35 diacetoxy 6 keto 22,23- bisnor 5m chol 7 en 24 oic acid methyl ester describedby Kerb et a1., Tetrahedron Letters (1966), 1387. Upon mild alkaline hydrolysis of the diacetate with dilute aqueous methanolic potassium carbonate there is obtained the free 2/3,3 3-diol, which, upon treatment with an aldehyde or ketone of the type conventionally employed in forming acetals and ketals, and particularly acetone, in the presence of p-toluenesulphonic acid yields the corresponding acetal or ketal and specifically the acetonide (I: R =keto; A=B=methyl).

Reduction of the thus formed 25,3/8-acetonide of the 26,35 dihydroxy 6 keto 22,23 bisnor 5a chol- 7-en 24 oic acid methyl ester with an alkali metal hydride such as sodium borohydride or lithium aluminum tri-t-butoxy hydride affords a mixture of 6c: and 6B-carbinols (I: R =hydroxy; A=B=methyl) with the 6p carbinol in predominant amount. The compounds may be separated through chromatography or subjected to the next step as a mixture.

Upon further reduction of the thus formed 2,8,3 8-isopropylidenedioxy 6 hydroxy 22,23 bisnor 5achol 7 en 24 oic acid methyl ester with an alkali metal hydride such as lithium aluminum hydride, there is obtained the C22 alcohol, i.e., 2fi,3B-isopropy1idenedioxy 6, 22-dihydroxy bisnor SOL-ch01 7 ene or 25, 3}?! isopropylidenedioxy 6 hydroxy 20 hydroxymethyl pregn-7-ene (II: R =hydroxy; A:B=methyl).

Reoxidation of the diol, for example, with ice-cold chromium trioxide in pyridine, rapidly yields the unsaturated 6-keto alcohol (II: R :keto; A=B-methyl), which, upon further oxidation with a mixed reagent comprising a dialkyl carbodiimide such as diethyl carbodiimide, a hydrocarbon sulfoxide such as dimethyl sulfoxide, a solvent such as benzene, in the presence of an acid catalyst such as pyridinium trifluoracetate, affords the 6-kcto-20-aldehyde (III: A=B=methyl).

For introduction of a hydroxy group at C-20, the carbonyl function of the aldehyde is utilized for the next step which is formation of the enol acylate (IV) by reaction of the aldehyde, preferably with boiling acetic anhydridezsodium acetate. The enol acetate is then epoxidized with a peracid such as m-chloroperbenzoic acid in a solvent such as benzene to produce approximately equal amounts of two C20,22 expoxide isomers (V). This mixture may be separated by conventional chromatographic techniques or alternatively, the process may be continued using the mixture of epoxides.

Upon treatment of the epoxides with dilute methanolic potassium carbonate under an atmosphere of nitrogen, there is obtained the stereoisomeric hydroxyaldehydes (VI). The mixture of hydroxyaldehydes are treated with dihydropyran and a catayltic amount of p-toluenesulphonic acid in dry benzene to obtain the corresponding isomeric tetrahydropyranyl ethers (VII and VIII).

Alternating, the hydroxyaldehydes (VI) are separated by conventional methods such as chromatography or fractional crystallization and pyranylated separately to each afford two diastereomeric pyranyl ethers differing only within the tetrahydropyran ring (VII and VIII).

Hydroxylation at C-14, if desired, is then effected by reacting the pyranyl ethers with selenium dioxide in an ether solvent such as dioxane to give the 14a-hydroxy form (IXa and IXb).

For introduction of the side chain at -22, alkylation is effected either prior to or subsequent to the hydroxylation at C-14, with 3-methyl-3-tetrahydropyran 2 yloxybut-l-yne in a solvent such as tetrahydrofuran at 20 C. to afford a mixture of epimeric C22 tetrahydropyranyl ethers (X, XI).

'Epimerization of the hydrogen at C-5, if desired, may be effected at this stage by treating the alkylated product with aqueous methanolic potassium carbonate.

The resulting products may be reduced to the olefinic stage or completely saturated either prior to or subsequent to the hydroxylation at C-l4 or prior to or subsequent to the epimerization at C5. Thus the product of the alkylation is catalytically reduced with Lindlar catalyst or palladium on charcoal for a period of time of the order of one-half hour to yield the corresponding 25, 3,8 alkylidenedioxy 14 hydroxy 20,22,25 tri(tetrahydropyran 2 yloxy) 5 cholest 7,23 dien 6- ones or 14 desoxy compounds, which, upon further hydrogenation in the presence of palladium, are transformed into 218,318 alkylidenedioxy 14a hydroxy 20, 22,25 tri(tetrahydropyran 2 yl oxy 5 cholest-7-en-ones or the corresponding -desoxy compounds. Separation of the C- and C-22 isomers may be performed at this stage although this may be effected before the previous step or after the subsequent step. Treatment of the thus reduced product with an acid such as hydrochloric acid, oxalic acid or formic acid for a period of time from about thirty minutes to about two hours results in the removal of the tetrahydropyranyl groups thus regenerating hydroxy groups, while prolonging this treatment to from about 10 to 20 hours cleaves, in addition, the 2B,3;3-alkylidenedioxy function thereby regenerating hydroxy groups in the 2 8 and 3,6 positions as well. There are thus formed the polyhydroxy steroids XII, XIII, XIV and XV including crustecdysone (XII: R =OH). Esters, if desired, may be produced by treatment with conventional acylating agents derived from hydrocarbon carboxylic acids of less than 12 carbon atoms.

While the foregoing transformations have been described in connection with the synthesis of crustecdysone and analogues thereof, they may also, with various modifications, be employed in the preparation of other valuable compounds as heretofore described. Accordingly by eliminating the epimerization of the C5 hydrogen, there are obtained upon execution of the other steps herein described, compounds bearing the Set-hydrogen. Similarly, elimination of the 14-hydroxylation step yields compounds bearing a I la-hydrogen. By oxidizing the C-22 hydroxy group prior to complete reduction of the C-23, 24 bond there are obtained compounds having a 22-carbonyl group in lieu of the 22-hydroxy derivatives. Further, 25-desoxy analogs are obtainable by substituting a bromomagnesium derivative of 3-methyl-butane for the 3-methyl-3-tetrahydropyran-Z-yloxy-but-l yne described hereinabove. Various combinations of these modifications are typified hereafter and others are apparent to those skilled in the art from the present disclosure of the invention.

The following examples serve to further typify the nature of the invention but, being presented solely for the purpose of illustration, these examples should not be construed as a limitation of the scope of this invention.

EXAMPLE I A solution containing 5 ml. of acetic acid, 2 ml. of acetic anhydride and 450 mg. of 2p,3fi-diacetoxy-5u-hydroxy-6-keto-22,23-bisnorchol-7-en 24 oic acid methyl ester and 100 mg. of p-toluenesulfonic acid is stirred at room temperature for 3.5 hours and then added cautiously to dilute aqueous potassium bicarbonate and subjected to extraction with methylene chloride. The methylene chloride extracts are thereafter washed with water and saturated sodium chloride solution and concentrated to dryness under vacuum, yielding 2fl,3fl,5a-triacetoxy-6- keto-22,23-bisnorchol-7-en-24-oic acid methyl ester, M.P. 184.5185 C., [11] +58.

To a solution of mg. of 2 8,3p,5a-triacetoxy-6-keto- 22,23-bisnorchol-7-en-24-oic acid methyl ester and 5 ml. of acetone is added 4 ml. of a 25% solution of chromous chloride in 1 N hydrochloric acid. The solution is allowed to stand for two minutes, whereupon it is poured into a waterzmethylene chloride mixture. The organic layer is separated and washed successively with dilute aqueous potassium bicarbonate, water, and saturated sodium chloride solution, dried over sodium sulfate, and concentrated to dryness, thus yielding 2fl,3,B-diacetoxy-6- keto-22,23-bisnor-5a-chol-7-en-24-oic acid methyl ester, described by A. Fustenmeier et al., Helv. Chim. Acta 49', 1591 (1966).

EXAMPLE II A mixture of 219,3,B-diacetoxy-6-keto-22,23-bisnor-5av EXAMPLE III A mixture containing 7.14 g. of 219,3,B-dihydroxy-6-keto- 22,23-bisnor-5m-chol-7-en-24-oic acid methyl ester, ml. of acetone, and 66 mg. of p-toluenesulfonic acid is stirred at room temperature for approximately 1 hour. The reaction mixture is thereafter poured into cold, saturated aqueous potassium bicarbonate solution, water is then added and the mixture is filtered, dried over sodium sulfate and evaporated to dryness to yield 7.42 g. of 218,33- isopropylidenedioxy-6-keto-22,23-bisnor-5a-chol 7 en- 24-oic acid methyl ester. The product was recrystallized from acetonezmethylene dichloride to afford 5.49 g. of pure product, M.P. 255-257"; [a] +19"; A 244 mp (e 12,780).

Analysis.Calcd. for C H O C, 72.52; H, 8.90; O, 18.58. Found: C, 72.85; H, 8.80; O, 18.53.

EXAMPLE IV A mixture of 5 g. of 2B,3fl-isopropylidenedioxy-6-keto- 22,23-bisnor-5u-chol-7-en-24-oic acid methyl ester and 300 ml. of anhydrous tetrahydrofuran are heated to reflux. To the mixture there is added 5.2 g. of lithium aluminum tri(t-butoxy)hydride and the mixture is stirred at reflux for about forty minutes. The reaction mixture is cooled,

5.2 ml. of acetic acid is added and then extracted with methylene dichloride and water. The extracts are concentrated to dryness under vacuum to yield 25,3[i-isopropylidenedioxy-6-hydroxy 23,24-bisnor-5a-chol-7-en-22-oic acid methyl ester which may be separated into the 60: and 66 isomers by fractional crystallization and silica chromatography. By recrystallization from methylene chloride, ether and hexane there were obtained 3.15 g. of 2fl,3 8-isopropylidenedioxy 613 hydroxy-22,23-bisnor-5achol-7-en-24-oic acid methyl ester, M.P. 214-216"; [a] 6.5; N.M.R. 67.5 (19-H), 105 (OH), 220250 C.P.S. mult. (6a-H).

Analysis.Calcd. for C -H C, 72.19; H, 9.32; O, 18.49. Found: C, 72.43; H, 9.13; O, 18.34.

EXAMPLE V A solution of 2 g. of 2,3,3fi-isopropylidenedioxy-6B- hydroxy-22,23-bisnor-5u-cho1-7en-24-oic acid methyl ester in 50 ml. of tetrahydrofuran is added over a 30- minute period to a stirred suspension of 200 mg. of lithium aluminum hydride in 20 ml. of tetrahydrofuran, maintaining a temperature of about 20 C. The mixture is stirred for 45 minutes and at the end of this time, the excess reagent is destroyed through the addition of 40 ml. of ethyl acetate followed by 28 ml. of water. After stirring for an additional 30 minutes, the mixture is filtered through Celite and the filtrate evaporated to dryness. The white solid is crystallized from methylene dichloridezhexane to give 1.51 g. of 2,8,3fi-isopropylidenedioxy-6,B-hydroxxy-20a-hydroxymethyl-pregn-7-ene, M.P. 242-244, [od 21.

Analysis.Calcd. for C H O C, 74.21; H, 9.97; O, 15.82. Found: C, 74.41; H, 10.09; 0, 15.66.

EXAMPLE VI A mixture of 10.6 g. of 2,6,3fl-isopropylidenedioxy-6fihydroxy-20a-hydroxymethyl-pregn-7-ene in 254 ml. pyridine at ice temperature is added to 8.5 mg. chromium trioxide in 130 m1. pyridine at ice temperature and stirred at ice temperature for 2 hours. Thereafter 100 ml. of methylene dichloride is added with Celite, the mixture is filtered, extratced with water, then ether until the color is removed, dried and concentrated to obtain g. of 2,8,36- isopropylidenedioxy 6-keto-20u-hydroxymethyl-pregn-7- ene, M.P. 258-260"; [a1 +10; k 245 mp. (e 13, 160).

Analysis.Calcd. for C H O C, 74.59; H, 9.52; O, 15.90. Found: C, 74.54; H, 9.66; O, 16.02.

EXAMPLE VII A solution of 150 g. sodium carbonate, 224 g. of sodium chloride, 1 l. of water is added to 132 g. diethylurea in 600 ml. methylene dichloride with stirring at a temperature below 27. While the temperature is maintained below 30 with ice cooling, 14 g. of cupric chloride is added. After stirring the mixture at room temperature for 4 hours, the reaction mixture is filtered and separated. The organic layer is washed with 200 ml. water, the aqueous layer is then washed three times with 200 ml. of methylene dichloride. The organic material is dried over sodium sulfate, concentrated under vacuum pressure. A fraction amounting to 50 g. of diethylcarbodii-mide boiling at 90-91 at 250 mm. is collected, diluted with 100 ml. dry benzene and the total volume of 152 ml. is stored at room temperature.

A mixture of 8 g. of 2,8,3 3-isopropylidenedioxy-20ahydroxymethyl-pregn-7-en-6-one, 300 ml. of dimethyl sulfoxide, 12.5 ml. of pyridine and 2.5 ml. of trifiuoroacetic acid is treated with 29.8 ml. of the diethylcarbodiimide-benzene mixture. After 5 hours, an additional 20 ml. of diethylcarbodiimide-benzene is added. After 11 hours an additional 1 ml. of trifluoroacetic acid in 5 ml. of pyridine are added and the mixture is allowed to stand for one and one-half hours. The solid which precipitated is filtered and washed with methanol. There 10 is obtained 5.95 g. of 2,8,3fi-isopropylidenedioxy-6-keto- 20a-formyl-pregn-7-ene, M.P. 250252; [111 +8 x 244 mu (6 13, 840), N.M.R. 39.5 (IS-H), 69 doublet, J 6. 5 (21-H), 577 doublet, 6 c.p.s. (20-CHO).

Analysis.Calcd. for C H O C, 74.96; H, 9.06; O, 15.98. Found: C, 74.93; H, 8.93; O, 16.11.

EXAMPLE VIII A mixture of 244 mg. of 2/3,3B-isopropylidenedioxy-6- keto-20u-formyl-pregn-7-ene dissolved in 25 ml. of acetic anhydride to which 440 mg. of sodium acetate is added is refluxed for 4 hours under an atmosphere of nitrogen. The mixture is then diluted with water, Washed with ice cold aqueous potassium bicarbonate and water. The solvent is evaporated and the acetic anhydride removed with benzene. The residue is chromatographed to give 135 mg. of the enol acetate, M.P. 185-189: u 1760, 1675, 1625 cmr N.M.R. me. 1.71 (21-H), 2.12 (OAc), 6.8 p.p.m. (22-H).

Analysis.Calcd. for C H O C, 73.27; H, 8.65. Found: C, 73.31; H, 8.90.

EXAMPLE IX A mixture of mg. of the enol acetate produced in the preceding example in 10 ml. of benzene is treated with 40 mg. of m-chloroperbenzoic acid. The mixture is allowed to stand overnight, and then is washed with 2% sodium hydroxide, then with water, sodium sulfite solution and again with water. The reaction mixture is then evaporated to dryness and the residue chromatographed to obtain 108 mg. of a crystalline mixture of two C-20 isomeric epoxides in approximately equal amounts, M.P. 190205; N.M.R. 100 me. 0.70, 0.80 (18-H), 1.50, 1.55 (2l-H), 2.09, 2.11 p.p.m. (OAc).

Analysis.-Calcd. for C H O C, 70.11; H, 8.35. Found: C, 70.23; H, 7.96.

EXAMPLE X A solution containing 100 mg. of the epoxides obtained in Example IX dissolved in. 40 ml. of a solution of potassium bicarbonate prepared from mg. of potassium bicarbonate in 20 ml. of water and ml. of methanol, was stirred at room temperature under nitrogen for one and one quarter hours. The reaction mixture was diluted with methylene dichloride and washed with water until the washings were neutral. The solution is then concentrated to yield 90 mg. of a mixture of isomers which is separated by thin layer chromatography to yield 26,35- isopropylidenedioxy 6 keto 20fl-hydroxy-20-formylpregn-7-ene, M.P. 228-231"; N.M.R. 100 me. 0.69 (IS-H), 1.36 (21-H), 9.61 p.p.m. (CHO).

Analysis.--Calcd. for C H O C, 72.08; H, 8.71. Found: C, 71.53; H, 8.87 and 25,3B-isopropylidenedioxy- 6-keto-20a-hydroxy-20-formyl-pregn-7-ene, M.P. 234- 237; N.M.R. me. 100 0.62 (18-H), 1.27 (2l-H), 9.62 p.p.m. (CHO).

Analysis.Calcd. for C H O C, 72.08; H, 8.71. Found: C, 72.13; H, 9.03.

EXAMPLE XI 270 mg. of 213,3fl-isopropylidenedioxy-6-keto-20a-hydroxy-20-formyl-pregn-7-ene is dissolved in 67 ml. of benzene. After distilling off 30 ml. of benzene, 0.68 ml. of dihydropyran and 12.8 ml. of a solution of 50 mg. of p-toluenesulfonic acid in 60 ml. benzene are added. The mixture is allowed to stand for 3 hours, then washed with saturated potassium bicarbonate solution, water and dried over sodium sulfate. Evaporation under reduced pressure affords a mixture which is resolved by chromatography to yield two diastereomeric pyranyl ethers of 25,3,8 isopropylidenedioxy 6-keto-20/3-tetrahydropyran- (2) yloxy 20 formyl pregn 7 ene differing only within the tetrahydropyran ring, one isomer having a M.P. 211.5215; N.M.R. (mc. 100) 0.85 (18-H), 1.04

11 (19-H), 1.34 (21-H), 4.65 (OCHO), 5.73 (7-H), 9.83 p.p.m. (CHO).

Analysis.Calcd. for C H O C, 71.97; H, 8.86. Found: C, 71.27; H, 8.86 M.S. 500 (M+), 287, 329, 85, and the other isomer having a M.P. 181-183, N.M.R. (mc. 100) 0.80 (18-H), 1.03 (19-H), 1.40 (21-H), 4.75 (CH), 5.74 (7-H), 9.72 p.p.m. (CHO).

Analysis.Calcd. for C H O' C, 71.97; H, 8.86. Found: C, 71.97; H, 8.86. M.S. 500 (M+), 387, 329, 85.

EXAMPLE XII 210 mg. of 2B,3/3-isopropylidenedioxy-6-keto-20 3-hydroxy-20-formyl-pregn-7-en is dissolved in 52 ml. of benzene. After 25 ml. of benzene is distilled otf, 0.68 ml. of dihydropropyran and ml. of a solution of 50 mg. of p-toluenesulfonic acid in 60 ml. benzene is added. The mixture is allowed to stand for 3 hours and is then worked up in the same manner as described in Example XI.

The mixture of isomers is resolved by thin layer chromatography to yield two diasteromeric pyranyl ethers of 26,3 8 i-sopropylidenedioxy 6 keto 2'05 hydroxy- -formyl-pregn-7-ene, differing only in the pyranyl ring with one having a M.P. 229-233"; N.M.R. (mc. 100) 0.70 (18-H), 1.02 (19-H), 1.33 (2l-H), 4.65 (OCHO), 5.71 (7-H), 9.73 p.p.m. (CHO).

Analysis.-Calcd. for C H O C, 71.97; H, 8.86. Found: C, 72.14; H, 8.72. M.S. 500 (M+), 387, 329, 85, and the other having a M.P. 225-228; N.M.R. (mc. 100) 0.70 (l8-H), 1.03 (19-H), 1.44 (2l-H), 4.69 (OCHO), 5.72 (7-H), 9.75 p.p.m. (CHO).

Analysis.Calcd. for C H O C, 71.97; H, 8.86. Found: C, 72.08; H, 8.84. M.S. 500 (M+) 387, 329, 85.

EXAMPLE XIII A mixture containing 120 mg. of 25,3fi-isopropy1idenedioxy 6 keto 20a tetrahydropyranyloxy-20-formylpregn-7-ene (the first isomer produced in Example XI), 17 ml. of dioxane and 45 mg. of selenium dioxide was stirred for two hours at 55. The reaction mixture is poured into dilute aqueous potassium bicarbonate and extracted with ethyl acetate. The extracts are washed with saturated sodium chloride solution until neutral, dried and filtered through Celite diatomaceous earth. The filtrate is concentrated to dryness, atfording 105 mg. of the 14u-hydroxy derivative M.P. 226-230"; N.M.R. (me. 100) 0.94 (18-H), 1.04 (19-H), 1.36 (21-H), 4.66 (OCHO), 5.89 (7-H), 9.86 p.p.m. (CHO).

Analysis.Calcd. for C H O C, 69.74; H, 8.58. Found: C, 69.90; H, 8.35.

EXAMPLE XIV A mixture containing mg. of selenium dioxide, 8

ml. of dioxane and 56 mg. of 2B,3/3-isopropylidenedioxy- 6 keto 20a tetrahydropyran-2-yloxy-pregn-7-en (the second isomer produced in Example XI), was treated in the same manner as set forth in Example XII to afiord the corresponding 14a-hydroxy derivative, N.M.R. (me. 100) 0.86 (18-H), 1.02 (19-H), 1.43 (21-H), 4.76 (OCHO), 5.92 (7-H), 9.66 p.p.m. (CHO).

EXAMPLE XV Analysis.Calcd. for C H O C, 69.74; H, 8.58. H

Found: C, 69.85; H, 8.18.

12 EXAMPLE XVI To 0.9 ml. 3-met'hyl-3-tetrahydropyran-2-yloxy-but-1- yne in 25 ml. tetrahydrofuran is added 1.3 ml. of a 3 N solution of methyl magnesium bromide in tetrahydrofuran under nitrogen and the mixture is stirred overnight at room temperature.

To 40 mg. of the product of Example XV dissolved in 3 m1. of tetrahydrofuran, there is added at ice temperature under nitrogen, 2.5 ml. of the Grignard reagent as prepared above. The reaction mixture is stirred for four and one-half hours at room temperature and then diluted with ethyl acetate, washed with an aqueous solution of potassium sodium tartrate, then with water to neutral and concentrated to dryness. Chromatography yielded 20 mg. of 2 8,318 isopropylidenedioxy-l4a-hydroxy 20a,22,25 tri(tetrahydropyran 2 yloxy)-5acholest-7-en-23-yn-6-one which upon further purification is separated into the 225 isomer, N.M.R. (mc. 0.80 (IS-H), 1.03 (19-H), 1.45 (21-H), 1.49, 1.57 (26-H, 27-H), 4.39 (22-H), 5.16 (OCHO), 5.92 p.p.m. (7-H). The 220: isomer is isolated similarly but in lesser quantity.

EXAMPLE XVII To 16 mg. of the product of Example XVI dissolved in 0.4 ml. of tetrahydrofuran there is added 2 ml. of 0.33% potassium carbonate in 10% aqueous methanol and the mixture is stirred overnight under nitrogen. The reaction mixture is then extracted with ethyl acetate, washed with water, dried over sodium sulfate and evaporated to dryness. By chromatography with ethyl acetatezhexane, there is obtained 10 mg. of the 5p isomer and 6 mg. of the starting 5a isomer. The recovered starting material is again equilibrated to yield an additional 4 mg. of the 5,8 isomer with recovery of 2 mg. of the 50: isomer.

EXAMPLE XVIII A mixture containing 14 mg. of 25,3,6-isopropylidenedioxy 14oz hydroxy 20a,22,25 tri(tetrahydropyran- 2-yloxy)-5a-cholest-7-en-23-yn-6-one, 9 ml. of N/20 HCl in 10% aqueous tetrahydrofuran is allowed to stand at room temperature for about three hours. The reaction mixture is then washed to neutrality with potassium bicarbonate and extracted with ethyl acetate. The combined ethyl acetate extracts are washed with water, dried over sodium sulfate, and concentrated to dryness to yield 213,35, 14m,20a,22,25 hexahydroxy 5oz cholest 7 en- 23-yn-6-one, which are separated into the 220: and 2213 isomers by silica chromatography.

Similarly, by substituting the 56 isomers of Example XVII in the above procedure, there are obtained the corresponding 2B,3B,14a,20a,22a,25 hexahydroxy 5B- cholest-7-en-23-yn-6-one and 25,35,l4u,20m,22p,25-hexahydroxy-5fl-cholest-7-en-23-yn-6-one.

EXAMPLE XIX In the same manner as described in Example XVI, 50 mg. of the product of Example X111 dissolved in 3 ml. tetrahydrofuran is reacted with 2.5 ml. of the Grignard reagent as prepared in Example XVI. The reaction mixture is washed with water, extracted with ethyl acetate, dried and evaporated to dryness. By chromatography the product was identified as 2p,3,3-isopropylidenedioxy-l4ahydroxy 2013,2235 tri(tetrahydropyran 2 yloxy)- 5a-cholest-7-en-23-yn-6-one, N.M.R. (mc. 100) 0.88 (IS-H), 1.04 (19-H), 1.37, 1.38, 1.57 (21-1-1, 26-H, 27-H), 5.11 (OCHO), 5.92 p.p.m. (7-H). The C-ZZa and C-22fl forms may be separated via chromatography over silica or may be used as a mixture of the two.

Without further purification, the crude product is dissolved in 0.5 ml. tetrahydrofuran to which is added 2 ml. of 0.33% potassium carbonate in 10% aqueous methanol. The mixture is kept overnight under nitrogen with stirring.

13 The mixture is then taken up in ethyl acetate:hexane and chromatographed to yield 29 mg. of the 8 isomer and 15 mg. of the starting 55 isomer. The latter is recycled to yield an additional mg. of the 5,8 isomer with 5 mg. of the 5 3 isomer recovered.

EXAMPLE XX A solution of 39 mg. of the 5 8 isomer produced in Example XIX in 10 ml. of ethanol is hydrogenated for one hour in the presence of 40 mg. of 5% poisoned palladium-on-calcium carbonate catalyst. At the end of this time, the reaction mixture is filtered and the filtrate evaporated to yield 35 mg. of crude 2fl,3;9-isopropylidenedioxy 140a hydroxy 2013,2235 tri(tetrahydropyran- 2-yloxy)-5 8-cholest-7,23 lien-done. The 220a and 22 8 forms may be separated by chromatography over silica gel in ethyl acetate-hexane system.

The foregoing mixture is stirred for nine hours with 3 ml. of N/ZO HCl in 10% aqueous tetrahydrofuran. The reaction mixture is then washed with aqueous saturated sodium chloride solution until neutral. The reaction mixture is then taken up in methanolzchloroform and chromatographed to yield 25,35,1411,203,22a,25-hexahydroxy- 5,8-cholest-7,23-dien-6-one and 2 8,3 8,14u,20fi,22B,25-hexahydroxy5 8-cholest-7,23-dien-6-one.

In a similar fashion the 5m isomer of Example XIX is transformed into the corresponding 5oz derivatives.

By substituting the product of Example XVI in the above described method, there are obtained 2 3,3;3-isopropylidenedioxy 14oz hydroxy 20a,22,25 tri(tetrahydropyran-Z-yloxy)-5a-cholest-7,23-dien-6-one and subsequently 2 3,3 9,14a,200t,22,25 hexahydroxy-Sa-cholest-7, 23-dien-6-one which can be separated into the 22a and 22B isomers.

EXAMPLE XXI By following the procedure of Example XVII the 2,8, 3/3 isopropylidenedioxy 14oz hydroxy 20a,22a,25 tri (tetrahydropropyran 2 yloxy-) 5a cholest 7 en 23- yn-6-0ne and 2,8,3,8-isopropylidenedioxy-14ot-hydroxy-20a, 223,25 tri(tetrahydropyran 2 yloxy-) 5a cholest 7- en-23-yn-6-one and the corresponding 5/3 isomers produced in Example XIX are transformed into the free hex-ahydroxy compounds, namely, 2B,3,B,14ot,20ot,220c,25- hexahydroxy-5a-cholest-7-en-23-yn-6-one; 2fi,3fi,14oz,200t, 22p,25-hexahydroxy-5a-cholest-7-en-23-yn-6-one and the corresponding 5,8 isomers.

In a similar fashion, the 25,35-isopropylidenedioxy-14ahydroxy 20cc,22,25 tri(tetrahydropyran 2 yloxy)- 5a-cholest-7,23-dien-6-one and the 5 8 isomer produced in Example XX are transformed into the free hexahydroxy compounds.

EXAMPLE XXII 14 mg. of the crude product produced in Example XVII dissolved in 10 ml. of ethanol is hydrogenated in the presence of 16 mg. of 5% poisoned palladium-on-calcium carbonate catalyst for one half hour. At the end of this time, the catalyst is filtered off to yield, after work up as set forth in Example XX, mg. of 2,3,3fi-isopropylidenedioxy 14a hydroxy 20oz,22,25 tri(tetrahydropyran-2- yloxy)-5[3-cholest-7,23-dien-6-one which can be separated by silica chromatography into the C-22a and C22/3 isomers.

The crude material dissolved in 15 ml. of ethanol is hydrogenated for three hours in the presence of 40 mg. of 5% palladium-on-charcoal catalyst and worked up as set forth in Example XX, except that the product is stirred for 5 hours with 5 ml. of N/ 10 hydrochloric acid in 10% aqueous tetrahydrofuran to afford 14 mg. of 2fi,3fi,14a, 20a,22B,25-hexahydroxy 5,8 cholest-7-en6-one (ecdysterone) N.M.R. (me. 100) 1.07 (19-H), 1.20 (18-H), 1.36 (26-H, 27-H), 1.56 (21-H), 6.17 (7-H), and 25,313, 14u,20a,22a,2S-hexahydroxy-5B-cholest-7-en-6-one.

14 EXAMPLE XXIII By subjecting the products of Example XI to the method of Example XVI there are obtained the diastereoisomers of 2p,3B-isopropylidenedioxy-20/3,22(a and [3),25- tri(tetrahydropyran 2 yloxy) 5 a cholest-7-en-23-yn- 6-one differing only in the ZO-tetrahydropyranyl ring. The mixture of isomers is then treated with acid as set forth in Example XVIII to yield the free pentahydroxy compounds 2,B,3/8,20 8,22(a and [3),25 pentahydroxy 5acholest-7-en-23-yn-6-one. By further reaction with methanolic potassium carbonate as set forth in Example XVII there are obtained the corresponding SB-isomers. The 220: and 22,3 forms may be separated via chromatography over silica eluting 1:1 hexanezacetone.

EXAMPLE XXIV Similarly the products of Example XII are subjected to the method of Example XVI to yield the diastereoisomers of 25,3,8-isopropylidenedioxy-20a,22(a and fi),25- tri(tetrahydropyran-Z-yloxy) 5oz ch0lest-7-en-yn-6-one which upon treatment with acid as set forth in Example XVIII are transformed in the free pentahydroxy compounds 2fl,3B,20u,22(a and p),25 pentahydroxy 5acholest-7-en-23-yn-6-one. By treating the thus formed product according to the method of Example XVII, there are obtained the corresponding 55 isomers.

EXAMPLE XXV By hydrogenating 2;8,3,8,20/8,22,25 pentahydroxy 5acholest-7-en-23-yn-6-one of Example XXIII according to the method of Example XXII, there are first obtained 2p,3fi,20fl,22(u and [3),25 pentahydroxy-5a-cholest-7,23- dien-20-one and then 213,35,205,22u,25-pentahydroxy-5acholest-7-en-6-one and 219,3 8,20/3,225,25-pentahydroxy-5acholest-7-en-6-one.

In a similar fashion, the 5,8 isomers of Example XXIII are transformed successively into the corresponding 55 isomers.

EXAMPLE XXVI By substituting 2 3,3,B,20u,22(a and fl),25-pentahydroxy- 50c cholest 7 en 22 yne 6 one and 28,35, 20a, 22(1); and B),25 pentahydroxy-5fl-cholest-7-en-22-yne-6- one in the method of Example XXII there are obtained the corresponding 2fi,3B,20ot,22(ot and B),25 pentahydroxy 5 a cholest-7,23-dien-20-ones; 2fl,3B,20a,22(a and 13),25-pentahydroxy-5u-cholest-7-en-20-ones and the corresponding 5/8 isomers.

EXAMPLE XXVII By hydrogenating the 213,3 3,14m,20a,22,25-hexahydroxy- 5a-cholest-7-en-23-yn-6-one of Example XVIII by the method of Example XX, there are obtained 2fi,3/3,14oc,20ot, 22,25-hexahydroxy-5a-cholest7,23-dien 6-ones which can be separated into the C-22a and C-22;& isomers. Upon further hydrogenation there are obtained 25,3,6,14u,20a, 22a,25-hexahydroxy-5a-cholest-7-en-6-one and 2B,3fi,14a, 20a,22/8,25-hexahydroxy-5a-cholest-7-en-6-one.

EXAMPLE XXVIII By substituting 2B,3}3,14u,20oz,22(a and fi),25-hexahydroxy-5a-cholest-7-en-23-yne-6 ones of Example XXI in the method of Example XXVII, there are obtained the corresponding 2/3,3fl,14a,20oc,22(ot and [3),25 hexahydroxy- 5a-cholest-7,23-dien-6-ones and then 2fl,3fi,l4a,20a,22a, 2S-hexahydroxy-Sa-cholest-7-en-6-one and 2fl,3fl,14ot,20oz, 2213,25-hexahydroxy 5oz cholest-7-en-6-one, identical to those produced in Example XX.

Similarly, the 5B isomers produced in Example XXI are converted into the corresponding 5,3 isomers, identical with those produced in Example )Q(.

EXAMPLE XXIX 20 mg. of 2,8,3fl-isopropylidenedioxy-14u-hydroxy-20u, 22,8,25-tri(tetrahydropyran 2 yloxy) 5B cholest-7,23- dien-6-one prepared in Example XXII, is treated with 20 15 ml. of N/40 HCl in 10% aqueous tetrahydrofuran for 2 hours. The mixture is diluted with tetrahydrofuran, washed neutral with a saturated aqueous solution of sodium chloride and concentrated to dryness to yield 25,3fi-isopropylidenedioxy 14u,20a,22B,25 tetrahydroxy-B-cholest-7, 23-dien-6-one.

In a similar manner, 2 8,3 8 isopropylidenedioxy 14ahydroxy 20a,22,25 tri(tetrahydropyran 2 yloxy)-5acholest-7-en-23-yne-6-one, the SB-isomers and 20/3 isomer thereof prepared in Examples XVI, XVII and XIX are transformed into the corresponding 25,3;8-isopropylidenedioxy 14a,20,22,25 tetrahydroxy-5-cholest-7-en-23-yne- 6-one compounds.

EXAMPLE XXX A mixture of 10 mg. of 2 8,3,8-isopropylidenedioxy-14a, a,22/3,25 tetrahydroxy-5/8-cholest-7,23-dien-6-one prepared in Example XXIX, in 1 ml. of chloroform and 0.01 of isopropanol is stirred under nitrogen with 100 mg. of active manganese dioxide for six hours. At the end of this time, the manganese dioxide is removed by centrifugation and the resultant supernatant is evaporated to yield 2,8,3 3 isopropylidenedioxy 14a,20a,25 trihydroxy-Sficholest-7,23-dien-6,22-dione.

EXAMPLE XXXI By substituting 218,318 isopropylidenedioxy-Z0u,22;9,25- trihydroxy 513 cholest en 7,23-dien-6-one in Example XXX, there is obtained 2/3,Bfi-isopropylidenedioxy-20a,25- dihydroxy-5 3-cholest-7,23-dien-6,22-dione.

EXAMPLE XXXII By following the procedure of Examples XXIX and XXX, except that 2 9,313 isopropylidenedioxy 14cc hydroxy-20a,22fi,25-tri (tetrahydropyran 2 yloxy) 50ccholest17,23 dien 6 one prepared in Example XXI is substituted therein, there are obtained 213,313-isopropylidC116di0Xy-14a,20oc,25 trihydroxy-Sa-cholest-7,23 dien- 6,22-dione and 2 3,35,l4a,20a,25-pentahydroxy-5a-cholest- 7,23-dien-6,22-dione.

EXAMPLE XXXIII By substituting the bromo-magnesio derivative of 3- methyl butane in the method of Example XVI, there is obtained 213,3fi-isopropylidenedioxy-1401,22 dihydroxy- 20a-tetrahydropyran-2-yIoxy-5a-cholest-7-en-6-one.

By subjecting the product to the method of Example XVIII, there is obtained the 2 3,313,14a,20a,22fi-pentahydroxy-5a-cholest-7-en-6-one which is converted into the 5/3-is0mer by the method of Example XVII.

Similarly, by employing the above method to the product of Examples XI, XII and XIII, there are obtained the corresponding -desoxy derivatives.

EXAMPLE XXXIV A solution containing 25 mg. of 2}9,313,14a,20a,22;3,25- hexahydroxy-5fi-cholest-7-en-23-yne 6 one in 1 ml. of pyridine is treated with 0.1 ml. of acetic anhydride at 5 C. and allowed to stand for 4 hours. The reaction mixture is then evaporated under vacuum. Silica chromatography of the product affords 25,3/3-diacetoxy-14a,20a,22;3,- ZS-tetrahydroxy-SB cholest-7en-23-yne-6-one in addition to the 2 3-monoacetate and 3fi-monoacetate and a lesser amount of 25,3[3-22fi-triacetate.

In a similar manner the free polyhydroxy compounds of Examples XX-XXVHI and XXXII are converted in the corresponding acetates.

By substituting propionic anhydride, and cyclopentyl' propionic anhydride for the acetic anhydride, there are obtained the corresponding propionates and cyclopentylpropionates.

16 What is claimed is: 1. Compounds of the formula:

2. A compound according to claim 1 wherein each of R and R is hydrogen, R is a-hydroxy and R is as therein defined.

3. A compound according to claim 2 wherein R is fi-hydrogen.

4. A compound according to claim 2 wherein R is a-hydrogen.

5. The compound according to claim 1, 2fi,3fi,l4a,20a,- 223,25-hexahydroxy-5fi-choIest-7-en-23-yn-6-one.

6. Compounds of the formula:

wherein R is a-hydrogen or fl-hydrogen; R is hydrogen or hydroxy; R and R are each hydrogen or a hydro carbon carboxylic acyl group of less than 12 carbon atoms, and R is keto,

7. A compound according to claim 6 wherein R is hydroxy; each of R and R is hydrogen; R is and R is as defined therein.

17 18 8. A compound according to claim 7 wherein R is 18. The compound according to claim 13; 25,313,200, ,B-hydrogen. 22/3-tetrahydroxy-5/3-cholest-7-en-6-one.

9. The compound according to claim 6, 2B,3fi,14ot,20a,- 19. Compounds of the formula: 25-pentahydroXy-5 3-cholest-7,23-dien-6,22-dione.

10. Compounds of the formula: 5

OH CHQ I CHO 0H l R3O wherein each of A and B is hydrogen or lower alkyl, R

R II is hydrogen or hydroxy and R is hydrogen or tetrahydropyran-2-yl. wherein R is a-hydrogen or B-hydrogen; R and R are The M00655 0f Producing a Y each hydrogen or a hydrocarbon carboxylic acyl group of Py 'y y)' Whlch comprlses reactless than 12 carbon atoms, or taken together, the group mg a 20 tetfahydfopyfan -Y Y- -y y- Y A pregn 7-en-6-one with 3-methyl-3-tetrahydropyran-2-yloxy-but-l-yne magnesium bromide. 25 21. The process of claim 20 wherein a compound of the 13 following formula: in which each of A and B is hydrogen or lower alkyl; and R is hydrogen or hydroxy. CHEM 11. A compound according to claim 10 wherein R is fl-hydrogen, each of R and R is hydrogen and R is 4 hydroxy. 12. A compound according to claim 10 wherein R is flhydrogen and each of R R and R is hydrogen. A R1 13. Compounds of the formula: 3 I

o B O OH OH 5 CH; 0

CH3 H 40 in which each of A and B is hydrogen or lower a l kyl; R 3 is hydrogen or hydroxy and R is tetrahydropyran-Z-yl is reacted to produce a compound of the following formula:

, OR RO i 0R5 g I K CH3 Ra CH3 0 R CH3 wherein R is a-hydrogen or ,B-hydrogen; R and R are each hydrogen or a hydrocarbon carboxylic acyl group of less than 12 carbon atoms, or taken together, the group A A X I ll C B o in which each of A and B is a hydrogen or lower alkyl; and 6 is hydrogen or hydroxy whereln A, B, R and R are as previously defined.

14. A compound according to claim 13 wherein each of R and R is hydrogen, R is hydroxy and R is as there- References C'ted in defined. UNITED STATES PATENTS 1115a A compound according to claim 14 wherein R is 3,354,152 11/1967 Edwards et a1. 260 239'55 19- y rogen.

16. A compound according to claim 13 wherein R is ELBER L ROBERTS, Primary Examiner fl-hydrogen and each of R R and R is hydrogen.

17. The compound according to claim 13; 2635,200 US. Cl. X.R. 225,25-pentahydroxy-513-cho1est-7-en-6-one. 260--397.2, 999

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 4&$1,926 Dated December- 2. 1969 Inventor(s) Ge 'h P A Huppi 8t 81 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

( 1) Col 1 line 5; Col 1, line 60', C01 2, line i, and

Col. 2 line 20: In each formula the substituent "B" should read 1 R (four occurrences) (2) Col 1 line 60: the substituent "0H should read (3) Col g lines 1 to 15, that portion of the formula reading OH OH CH3 CH3 H OH should read H OH Col 2 lines +3 to 46, that portion reading:

OH -OH 43}! H \H ,\H H should read QH Page 1 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent i8 926 (Qontlnuedl Dated December 2, 1969 Inventor) Gerhard A Huppi et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

(5) Col. 6, in formula xv, that portion reading OH OH CH3 should read CH3 3 (6) C01. 11 line A, "287" should read 387 (7) Col. 15, line 28, "oholest 17,23" should read (molest-7,23

(8) Col. 16, lines 3 to 15 and 41 to 55, that portion of the formulae reading "R should read l (9) Col 16 lines 65 to 68, the group:

/ OH I ,/OH

\ should read Page 2 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 181 ,926 Lcontinued) Dated December 2, 1969 Invent0r(s) Gerhard A Huppi et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

(10) C01. 17, lines 5 to 18, the formula should appear as shown below instead of as in the patent:

(11) C01. 18, line 21, "7-on-6-one" should read 7-en-23-yn-6-one (12) C01. 18 line 22, delete "2-yloxy-", first occurrence (15) C01. 18, lines 27 to 38 and +3 to 56, that portion of the formulae reading:

l should read I H u 0 Papze 3 SIGNED AND .QEALEF' N0! 1 1m (SEAL) Anna:

M Fletdml H mm 1:. swim, JR

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